JP2011506328A - Co-administration of drugs linked to internalization peptides and anti-inflammatory agents - Google Patents

Abstract

The present invention provides a method of delivering a pharmacological agent linked to an internalizing peptide, wherein the inflammatory response inducible by the internalizing peptide is co-administered with an anti-inflammatory agent or biotin or a similar molecule is Inhibited by linking to internalization peptide. Such a method involves the administration of pharmacological agents linked to tat at high doses, in close proximity to inflammatory responses (mast cell degranulation, histamine release, and typical sequelae of histamine release (eg: Redundancy, fever, swelling, and hypotension)) are partly premised on the results described in the examples.
[Selection] Figure 1

Description

  This application claims priority from US Provisional Application No. 60 / 992,678, filed Dec. 5, 2007, the entire contents of which are hereby incorporated by reference.

  <Reference of “Sequence List”> The sequence list created on November 18, 2008 and provided in the file of SEQLIST026373000910US.txt having a capacity of 16,385 bytes is incorporated herein by reference in its entirety. The

  BACKGROUND OF THE INVENTION Many drugs are taken up by or through cells and / or taken up by cellular organelles to reach their intended therapeutic targets. Many larger molecules and some small molecules have limited ability to cross cell membranes by their own forces. The ability to cross the cell membrane can be increased by linking a pharmacological agent to an internalizing peptide (also known as a protein transduction domain, or membrane permeation domain). These peptides include tat, antennapedia peptides, and arginine rich peptides. These peptides are short basic peptides that are present in many intracellular and viral proteins and serve to mediate membrane permeation. A common feature of these peptides is that they are very cationic. Such peptides have been reported to promote the uptake of many different peptides and proteins into cells, as well as oligonucleotides, peptide nucleic acids and small molecules and nanoparticles. Uptake into cells and organelles and passage through the blood brain barrier has been reported.

  As one of the applications of internalization peptides, tat peptides were linked to peptide inhibitors of the interaction between post-synaptic thickening-95 protein (PSD-95) and NMDARs (Aarts et al., Science 298, 846). -850 (2002). The resulting chimeric peptide was tested in animal models of cells and stroke. The chimeric peptide was found to be taken up by neurons and reduce ischemic brain damage in the animal model. This suggests that PSD-95 / NMDAR peptide antagonists linked to internalization peptides can be used to treat stroke and other diseases (mediated by excitotoxicity). It came.

  Summary of the Invention The present invention provides a method of delivering a pharmacological agent to a subject. The method comprises administering to the subject the pharmacological agent linked to an internalizing peptide, and administering an anti-inflammatory agent to the subject, wherein the anti-inflammatory agent is the endogenous protein. Inhibiting the inflammatory response induced by the peptide. Optionally, the anti-inflammatory agent is an antihistamine or a corticosteroid. Optionally, the internalization peptide is a tat peptide. Optionally, the tat peptide has an amino acid sequence comprising GRKKRRQRRR (SEQ ID NO: 1), YGRKKRRQRRR (SEQ ID NO: 2), FGRKKRRQRRR (SEQ ID NO: 3), or GRKKRRQRRRPQ (SEQ ID NO: 4). Optionally, the pharmacological agent is a peptide. Optionally, the pharmacological agent is KLSSIESDV (SEQ ID NO: 5).

  The invention also provides the use of an anti-inflammatory agent in the manufacture of a medicament for inhibiting the inflammatory response induced by an internalizing peptide linked to a pharmacological agent.

  The present invention also provides a kit comprising a pharmacological agent linked to an internalization peptide and an anti-inflammatory agent that inhibits an inflammatory response induced by the internalization peptide.

  The present invention also provides the internalization peptide linked to the biotin with a reduced ability to induce an inflammatory response compared to an internalization peptide without biotin.

  The present invention also provides a method of delivering a pharmacological agent to a subject, wherein the method comprises administering to the subject the pharmacological agent linked to an internalization peptide, wherein the internalization peptide is biotinylated. And the biotinylation is a method of reducing the ability of the internalization peptide to induce an inflammatory response compared to the internalization peptide without the biotin.

  The present invention also provides a method for effecting treatment or prevention of diseases mediated by excitotoxicity, wherein the method is effective in the treatment regimen for effecting treatment or prevention of said disease, wherein PSD95 and NDMAR2B Administering a pharmacological agent that inhibits binding to an internalizing peptide to a subject having or at risk for the disease, and administering an anti-inflammatory agent to the subject. , Whereby the anti-inflammatory agent inhibits the inflammatory response induced by the internalizing peptide. Optionally, the pharmacological agent is a NMDAR receptor PL peptide. Optionally, the internalization peptide is a tat peptide. Optionally, the internalization peptide has an amino acid sequence comprising GRKKRRQRRR (SEQ ID NO: 1), YGRKKRRQRRR (SEQ ID NO: 2), FGRKKRRQRRR (SEQ ID NO: 3), or GRKKRRQRRRPQ (SEQ ID NO: 4). Optionally, the subject is a female. Optionally, the disease is stroke. In some methods, the subject is at risk for a transient cerebral ischemic attack as a result of undergoing cardiac surgery.

  The present invention further provides a method for effecting treatment or prevention of diseases mediated by excitotoxicity, wherein the method is effective in the treatment regimen for effecting treatment or prevention of said disease, wherein PSD95 and NDMAR2B Administering to a subject having or at risk the disease a pharmacological agent that inhibits binding to the internalization peptide, wherein the internalization peptide is biotinylated, Reduces the ability of the internalizing peptide to induce an inflammatory response.

  The present invention further provides a method for effecting treatment or prevention of diseases mediated by excitotoxicity, wherein the method is effective in the treatment regimen for effecting treatment or prevention of said disease, wherein PSD95 and NDMAR2B Administration of a pharmacological agent that inhibits binding to an internalizing peptide to a female subject having or at risk of said disease. Optionally, the internalization peptide is a tat peptide.

  The present invention further provides an improved method for delivering a pharmacological agent linked to an internalizing peptide to a subject, wherein the internalizing peptide is biotinylated or an inflammatory response induced by the internalizing peptide Or administered with an immunosuppressive agent that inhibits Optionally, the internalization peptide is a tat peptide.

  The present invention further provides a method of inhibiting an inflammatory response, said method comprising administering an anti-inflammatory agent to a subject who has been or will be administered a pharmacological agent linked to an internalizing peptide. In the method, whereby the anti-inflammatory agent inhibits the inflammatory response induced by the internalizing peptide.

  The present invention further provides a method of delivering a pharmacological agent to a subject, the method comprising administering to the subject the pharmacological agent linked to an internalizing peptide comprising: The subject is administered or will be administered an anti-inflammatory agent, whereby the anti-inflammatory agent inhibits the inflammatory response induced by the internalizing peptide.

It is a figure which shows the sex difference of the infarct size in a rat stroke P3VO model. Saline male: male stroke rat treated with saline (control). NA1 male: male stroke rat treated with Tat-NR2B9c (ie, peptide YGRKKRRQRRRKLSSIESDV (SEQ ID NO: 6) containing both the Tat sequence and the nine carboxy-terminal amino acids of the NR2B subunit). Saline female: female stroke rat treated with saline (control). NA1 female: female stroke rat treated with Tat-NR2B9c (ie, peptide YGRKKRRQRRRKLSSIESDV (SEQ ID NO: 6) containing both the Tat sequence and the nine carboxy-terminal amino acids of the NR2B subunit). Y axis: Infarct size. Measured relative to infarct size in male rats treated with saline alone (expressed as a percentage).

It is a figure which shows that the peptide containing a Tat sequence causes mast cell degranulation. CI: Calcium ionophore (positive control). NA-1: Tat-NR2B9c (ie, peptide YGRKKRRQRRRKLSSIESDV (SEQ ID NO: 6) containing both the Tat sequence and the nine carboxy terminal amino acids of the NR2B subunit). NR2B9c: Peptide KLSSIESDV (SEQ ID NO: 5); PSD-95 binding sequence (not including Tat sequence) of NMDA NR2B subunit. AA: Peptide YGRKKRRQRRRKLSSIE A D A (SEQ ID NO: 7); except that it can not bind to PSD-95 has a point mutation of two locations in the PSD-95 binding domain is identical to Tat-NR2B9c. Degranulation was measured by relative tryptase activity (% of control). Bars represent the mean ± SD of 3-6 independent experiments.

It is a figure which shows that mast cell degranulation by the peptide containing a Tat sequence is dose-dependent. CI: Calcium ionophore (positive control). NA-1: Tat-NR2B9c (ie, peptide YGRKKRRQRRRKLSSIESDV (SEQ ID NO: 6) containing both the Tat sequence and the nine carboxy terminal amino acids of the NR2B subunit). AA: Peptide YGRKKRRQRRRKLSSIE A D A (SEQ ID NO: 7); except that it can not bind to PSD-95 has a point mutation of two locations in the PSD-95 binding domain is identical to Tat-NR2B9c.

It is a figure which shows the mast cell degranulation by the peptide containing a Tat sequence variant. CI: Calcium ionophore (positive control). Tat-NR2B9c: Peptide YGRKKRRQRRRKLSSIESDV (SEQ ID NO: 6) containing both the Tat sequence and the nine carboxy terminal amino acids of the NR2B subunit. TAT: Tat peptide sequence YGRKKRRQRRR (SEQ ID NO: 2). 2B9c: Peptide KLSSIESDV (SEQ ID NO: 5); PSD-95 binding sequence of NMDA NR2B subunit (not including Tat sequence). AA: Peptide YGRKKRRQRRRKLSSIE A D A (SEQ ID NO: 7); except that it can not bind to PSD-95 has a point mutation of two locations in the PSD-95 binding domain is identical to Tat-NR2B9c. F-Tat-NR2B9c: Peptide FGRKKRRQRRRKLSSIESDV (SEQ ID NO: 8). Tat-NR2B9c K> A: YGRKKRRQRRRALSSIESDV (SEQ ID NO: 9). F-Tat-NR2B9c K> A: FGRKKRRQRRRALSSIESDV (SEQ ID NO: 10).

FIG. 3 shows that a peptide conjugate containing a Tat sequence cannot induce mast cell degranulation.

FIG. 6 shows the observed decrease in blood pressure observed after administration of 50 mg / kg Tat-NR2B9c to beagle dogs.

<Detailed Description>< Definition > A “chimeric peptide” means a peptide in which two component peptides that do not naturally associate with each other are linked to each other as a fusion protein or by chemical linkage.

  A “fusion” protein or polypeptide refers to a composite polypeptide, ie, two (or more) different heterologous polypeptides that refer to one contiguous amino acid sequence and are usually not fused to one polypeptide sequence. A composite polypeptide composed of the sequence

  The term “PDZ domain” refers to a modular protein domain of about 90 amino acids, including brain synaptic protein PSD-95, Drosophila septal junction protein Discs-Large (DLG), and epithelial tight junction protein ZO1 (ZO1). Characterized by having significant sequence identity (eg, at least 60%). PDZ domains are also known as Discs-Large homology repeats (“DHRs”) and GLGF repeats. PDZ domains generally appear to maintain a core consensus sequence (Doyle, D.A., 1996, Cell 85: 1067-76). Exemplary PDZ domain containing proteins and PDZ domain sequences are disclosed in US patent application Ser. No. 10 / 714,537, which is hereby incorporated by reference in its entirety.

  The term “PL protein” or “PDZ ligand protein” is a natural protein that forms a molecular complex with a PDZ domain, or when expressed separately from a full-length protein (eg, a peptide fragment of 3 to 25 residues, Refers to a protein whose carboxy terminus forms such a molecular complex (eg, as a peptide fragment of 3, 4, 5, 8, 9, 10, 12, 14, or 16 residues). The molecular complex can be observed in vitro using, for example, the “A assay” or “G assay” described in US patent application Ser. No. 10 / 714,537, or in vivo.

  The term “NMDA receptor” or “NMDAR” refers to a membrane bound protein known to interact with NMDA. The term thus includes the various subunit forms described herein. Such receptors may be derived from human or non-human (eg, mouse, rat, rabbit, monkey).

  “PL motif” is the amino acid sequence at the C-terminal of a PL protein (eg, C-terminal 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, or 25 consecutive residues) ("C-terminal PL sequence") or an internal sequence known to bind to the PDZ domain ("internal PL sequence").

  A “PL peptide” is a peptide comprising or consisting of, or otherwise based on a PL motif that specifically binds to a PDZ domain.

  The term “isolated” or “purified” refers to a contaminant in which the species of interest (eg, peptide) is present in a sample (eg, a sample obtained from a natural source containing the species of interest). It means that it has been purified from. Once the species of interest is isolated or purified, the species of interest becomes the major macromolecular (eg, polypeptide) species present in the sample. That is, the species is included on a molar basis in the composition more than any other individual species. Preferably, the species of interest comprises at least about 50% of all macromolecular species present on a molar basis. In general, an isolated, purified or substantially pure composition comprises more than 80 to 90 percent of all macromolecular species present in the composition. Most preferably, the species of interest is substantially homogeneously purified (ie, contaminated species cannot be detected in the composition by conventional detection methods) and the composition is substantially single. Made of high molecular species. The term isolated or purified does not necessarily exclude the presence of other components that are intended to work in combination with an isolated species. For example, an internalization peptide can be described as isolated despite being linked to an active peptide.

  "Peptidomimetic" refers to a synthetic chemical compound that is substantially identical in structure and / or functional properties to a peptide composed of natural amino acids. The peptidomimetic can include fully synthesized non-natural amino acid analogs, or can be a chimeric molecule having partially natural peptide amino acids and partially non-natural amino acid analogs. The peptidomimetic may also incorporate such conservative substitutions with any amount of natural amino acids, as long as the conservative substitutions do not substantially alter the mimetic's structure and / or inhibition or binding activity. Polypeptide mimetic compositions can include any combination of non-natural structural components, and typically consist of three structural groups: a) residues other than natural amide bond ("peptide bond") linkages Linkage group; b) non-natural residue instead of natural amino acid residue; or c) residue that induces mimicry of secondary structure, ie secondary structure (eg beta turn, gamma turn, beta sheet, alpha Helix structure etc.) that induces or stabilizes. In a peptidomimetic of a chimeric peptide comprising an active peptide and an internalization peptide, the active part or its internalization part or both may be a peptidomimetic.

  The term “specific binding” refers to the binding between two molecules, eg, a ligand and a receptor, in the presence of many other different molecules with another specific molecule (receptor). Characterized by the ability of a molecule (ligand) to bind (ie, showing that one molecule specifically binds to another molecule in a heterogeneous mixture of molecules). Specific binding of the ligand to the receptor is evidenced by a decrease in binding of the detectably labeled ligand to the receptor in the presence of an excess of unlabeled ligand (ie, in a binding competition assay). .

  Excitotoxicity is a pathological process in which neurons are damaged and killed by overactivation of the excitatory neurotransmitter glutamate receptor (eg, NMDA receptor (eg, NMDAR2B)).

The term “subject” includes humans and veterinary animals (eg, mammals).

  The term “drug” includes any element, compound, or entity, including but not limited to, for example, a pharmaceutical, therapeutic, pharmacological, medicated cosmetic, drug, toxin, natural Product, synthetic compound, or chemical compound.

  The term “pharmacological agent” means an agent having pharmacological activity. The drug includes a known drug and a compound that has been confirmed to have a pharmacological activity but is further evaluated for a therapeutic method. A chimeric agent includes a pharmacological agent linked to an internalizing peptide. An agent can be described as having pharmacological activity if it exhibits activity in a screening system that indicates that the active agent is useful or may be useful in the prevention or treatment of disease. The screening system can be in vitro, in cells, animals, or humans. The agent may be described as having pharmacological activity, regardless of whether further testing may need to establish actual prophylactic or therapeutic utility in the treatment of the disease.

  A tat peptide refers to a peptide comprising or consisting of GRKKRRQRRR (SEQ ID NO: 1), in a sequence that retains the ability to facilitate the uptake of peptides or other drugs linked thereto into cells. Residues not exceeding 5 residues are deleted, replaced or inserted. Preferably any amino acid change is a conservative substitution. Preferably any substitutions, deletions, or internal insertions in the assembly will leave the peptide having a net cationic charge, preferably similar to that of the aforementioned sequence. The amino acids of the tat peptide may be derivatized with biotin or similar molecules to reduce the inflammatory response, as described further below.

  Co-administration of a pharmacological agent linked to an internalizing peptide and an anti-inflammatory agent is an inflammation in which the two agents are administered within a sufficiently close time and the anti-inflammatory agent is induced by the internalizing peptide It means that the reaction can be inhibited.

  Statistically significant refers to a p-value that is <0.05, preferably <0.01, and most preferably <0.001.

< I. General > The present invention provides a method for delivering a pharmacological agent linked to an internalizing peptide, wherein the inflammatory response induced by the internalizing peptide is co-administered with an anti-inflammatory agent. Inhibition by administration or by linking the internalization peptide to biotin or similar molecules. Such a method is based in part on the results described in the examples, in which mast cell degranulation, histamine release is achieved by administering a high dose of a pharmacological agent linked to tat. And inflammatory reactions including the typical sequelae of histamine release (redness, fever, swelling, and hypotension) follow closely. Although implementation of the method of the present invention does not depend on an understanding of the mechanism, mast cell degranulation is a direct interaction between a cationic tat peptide and mast cells rather than being induced by an IgE antibody response. It is believed to be induced by The inflammatory response can be inhibited by co-administration of an anti-inflammatory agent and a pharmacological agent linked to tat or other internalizing peptide. A variety of widely used anti-inflammatory agents are suitable, including antihistamines and corticosteroids. Instead, the inventors have found that the ability of internalizing peptides to induce an inflammatory response can be reduced by linking them to biotin or similar molecules.

  The present invention further provides a method of treating or preventing a disease characterized by excitotoxicity (eg, stroke). Such diseases can be treated using a pharmacological agent linked to an internalizing peptide that inhibits the interaction between NMDARs and post-synaptic thickening 95 protein. Preferably, in such methods, the pharmacological agent is co-administered with an anti-inflammatory agent to inhibit the immune response induced by the internalizing peptide, or for the reasons described above, the internalizing peptide is biotinylated. Or linked to similar molecules. Regardless of whether anti-inflammatory agents or biotinylated internalization peptides are used in such methods, the treatment or prevention may be administered to both male and female subjects. Administration to female subjects is premised in part on the results described in the Examples where the treatment was found to be at least as effective as males in the rat model of stroke. The feasibility of administering to a female subject a pharmacological agent that inhibits the interaction between PSD95 and NMDAR has been found that inhibitors of nNOS are ineffective in treating excitotoxic disease in female subjects Contrast with previous results. Administration of nNOS inhibitors was reported to protect the damaging effects of stroke in male rats in the MCAO model, but increase cytotoxicity in female rats. Outside McCullough, Journal of Cerebral Blood Flow & Metabolism, 25: 502-512 (2005).

< II. Pharmacological Drug > The internalization peptide is linked to an arbitrary pharmacological drug, and takes up the drug through the cell membrane, the inner membrane (eg, nuclear membrane), and / or the blood brain barrier. Can be promoted. By conjugating the internalization peptide to a pharmacological agent, the bioavailability at the intended site is improved compared to the use of the pharmacological agent alone. Increased delivery due to the conjugated internalization peptide reduces the dose of pharmacological agents, effectively targets specific cell compartments (eg, the nucleus), and / or reduces toxicity due to the use of low doses. enable.

  Internalization peptides are particularly useful for pharmacological agents that need to enter cells and / or nuclei. Pharmacological agents with poor bioavailability, high doses or short half-lives, or neuroactive drugs that need to cross the blood brain barrier to exert activity are particularly suitable for binding internalized peptides Yes. Peptides are one type of pharmacological agent that is susceptible to internalization, for example through the use of peptide bonds that result in chimeric peptides comprising an amino acid sequence derived from a pharmacological agent and the amino acid sequence of the internalization peptide. Nucleic acids, and small organic molecules (less than 500 Da) are other examples of compounds that can be linked to internalization peptides.

Some guidance on pharmacological drug selection, conjugation methods and uses thereof are provided by the scientific and patent literature relating to internalization peptides (eg tat) (US Pat. No. 6,316,003 and US Pat. No. 5,804,604). See the specification). The following table lists the names of pharmacological drugs (some of which are approved drugs), diseases for which the drugs are useful for treatment, whether the disease is acute or chronic, and administration of drugs (within established limits) Lists comments on problems with existing drugs that can be partially overcome by the improved membrane permeation imparted by the pathway and internalization peptides.

One class of drugs of particular interest inhibits the interaction between PSD-95 and one or more NMDARs. Such agents are useful in reducing the damaging effects of stroke and other neurological conditions, at least in part mediated by NMDAR excitotoxicity. Such agents include peptides that contain or are based on the PL motif of the NMDA receptor or the PDZ domain of PSD95. Such peptides can also inhibit the interaction of PSD-95 with nNOS and other glutamate receptors (eg, kainite receptor or AMPA receptor). Preferred peptides are PDZ domains 1 and 2 of post-synaptic thickening 95 protein (PSD-95) (human amino acid sequence provided by Stathakism, Genomics 44 (1): 71-82 (1997)), one Or the C-terminal PL sequence of multiple NMDA receptor 2 subunits (including the NR2B subunit of the neuronal N-methyl-D-aspartate receptor (Mandich et al., Genomics 22, 216-8 (1994))) Inhibits interaction. NMDAR2B has GenBank number: 4099612, C-terminal 20 amino acids FNGSSNGHVYEKLSSIESDV (SEQ ID NO: 11) and PL motif ESDV (SEQ ID NO: 12). Preferred peptides inhibit human PSD-95 and the human NMDAR receptor. However, inhibition can also be shown from species variants of the protein. A list of NMDA and glutamate receptors that can be used is displayed below.

  Some peptides inhibit the interaction of PSD-95 with multiple NMDAR subunits. In such instances, the use of the peptide does not necessarily require an understanding of the contribution of each of the various NMDARs to excitatory neurotransmission. Other peptides are specific for a single NMDAR.

  The peptide can have an amino acid sequence comprising [S / T] -X- [V / L] comprising or based on a PL motif from the C-terminus of any of the above subunits. This sequence is preferably at the C-terminus of the peptides of the invention. A preferred peptide has an amino acid sequence comprising [E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L] (SEQ ID NO: 38) at the C-terminus. . Exemplary peptides are ESDV (SEQ ID NO: 12), ESEV (SEQ ID NO: 29), ETDV (SEQ ID NO: 39), ETV (SEQ ID NO: 40), DTDV (SEQ ID NO: 41), and DTEV (SEQ ID NO: 41). No. 42) as the C-terminal amino acid. Two particularly preferred peptides are KLSSIESDV (SEQ ID NO: 5) and KLSSIETDV (SEQ ID NO: 43). Such peptides usually have 3 to 25 amino acids (without internalization peptide), with peptide lengths of 5 to 10 amino acids, in particular 9 amino acids (also without internalization peptide) being preferred. In some such peptides, all amino acids consist of the C-terminus of the NMDA receptor (not including amino acids from the internalization peptide).

  Other peptides that inhibit the interaction between PDS95 and NDMARs include any PDZ domain 1 and / or 2 of PSD-95, or any that inhibits the interaction between PSD-95 and the NMDA receptor (eg, NMDA2B) Contains peptide subfragments. Such active peptides comprise at least 50, 60, 70, 80, or 90 amino acids from PDZ domain 1 and / or PDZ domain 2 of PSD-95. It is about amino acids 65-248 of PSD-95 provided by Stathakism, Genomics 44 (l): 71-82 (1997) (human sequence), or NP_031890.1, GI: 6681195 (mouse sequence), or others In the region corresponding to the amino acid of PSD-95.

  The appropriate pharmacological activity of a peptide, peptidomimetic or other agent can be confirmed using the animal model described in the Examples, if desired. Optionally, the peptide or peptidomimetic has the ability to inhibit the interaction between PSD-95 and NMDAR2B using, for example, the assay described in US Patent Application Publication No. 20050059597, which is incorporated by reference. Can also be screened. Useful peptides typically have an IC50 value of less than 5OμM, 25μM, 10μM, 0.1μM or 0.01μM in such assays. Preferred peptides usually have an IC50 value of 0.001 to 1 μM, more preferably 0.05 to 0.5 or 0.05 to 0.1 μM.

  These peptides, as described immediately above, are optional to improve the binding affinity of the inhibitor, to improve the ability of the inhibitor to be transported across the cell membrane, or to improve stability. However, it can be derivatized (eg, acetylated, phosphorylated, and / or glycosylated). As a specific example, if the third residue from the C-terminus is an inhibitor of S or T, this residue may be phosphorylated before using the peptide.

  Pharmacological agents also include small molecules that inhibit the interaction between PSD95 and NMDAR2B and / or other interactions described above. Suitable small molecule inhibitors are described in co-pending international application PCT / US2006 / 062715 and US patent application 60 / 947,892 filed July 3, 2007, each of which The whole is cited by reference. These molecules were identified by in silico screening of compound libraries that bind to PSD95, and binding of exemplary compounds was experimentally confirmed.

A number of suitable compounds are described in US Provisional Patent Application No. 60 / 947,883, the entire contents of which are hereby incorporated by reference. An exemplary class of suitable compounds is of the formula:
(In the formula, R ¹ is cyclohexyl substituted with 0-4R ^7, phenyl substituted with _{^{0-4R 7, - (CH 2)}} U - (CHR 8 R 9), branched C _1-6 alkyl ( A member selected from the group consisting of isopropyl, isobutyl, 1-isopropyl-2-methyl-butyl, 1-ethyl-propyl), and —NH—C (O) — (CR ¹⁰ R ¹¹ ) _V H;
Each R ⁷ is independently selected from the group consisting of C _1-6 alkyl, C _1-6 alkoxy, —C (O) R ¹² , OH, COOH, —NO, N-substituted indoline, and a cell membrane permeable peptide. A member;
Each R ⁸ and R ⁹ is independently selected from the group consisting of H, OH, cyclohexane, cyclopentane, phenyl, substituted phenyl, and cyclopentadiene;
Each R ¹⁰ and R ¹¹ is independently selected from the group consisting of H, cyclohexane, phenyl, and a cell membrane penetrating peptide;
R ¹² is selected from the group consisting of C _1-6 alkyl and aryl;
And each of u and v is independently 0 to 20;
Wherein one of R ² , R ³ , R ⁴ , R ⁵ and R ⁶ is —COOH, wherein the rest of R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are each independently Selected from the group consisting of F, H, OCH ₃ , and CH ₃ . )

One such compound is 0620-0057, whose structure is:

< III. Internalization Peptides > Internalization peptides are a well-known class of relatively short peptides that allow many cellular or viral proteins to permeate the membrane. Examples include antennapedia protein (Bonfanti, Cancer Res. 57, 1442-6 (1997)) (and variants thereof), human immunodeficiency virus tat protein, protein VP22, herpes simplex virus type 1 UL49 gene product, Penetratin, SynB1 and 3, transportan, Amphipathic, gp41NLS, polyarginine, and some plant and bacterial protein toxins (eg, ricin, abrin, modeccin, diphtheria toxin, Cholera toxin, anthrax toxin, heat labile toxin and Pseudomonas aeruginosa exotoxin A (ETA)). Other examples are described in the following literature (Temsamani, Drug Discovery Today, 9 (23): 1012-1019, 2004; De Coupade, Biochem J., 390: 407-418, 2005; Saalik Bioconjugate Chem. 15 : 1246-1253, 2004; Zhao, Medicinal Research Reviews 24 (1): 1-12, 2004; Deshayes, Cellular and Molecular Life Sciences 62: 1839-49, 2005) (all references are cited by reference).

A preferred internalization peptide is HIV virus tat. The tat peptide reported in previous studies contains or consists of the standard amino acid sequence YGRKKRRQRRR (SEQ ID NO: 2) found in HIV Tat protein. If there is an additional residue (adjacent to the pharmacological agent) that is added adjacent to such a tat motif, the residue is, for example, a natural residue that is added adjacent to this segment of the tat protein. Linker amino acids commonly used to join amino acids, spacers, or two peptide domains (eg, gly (ser) ₄ (SEQ ID NO: 44), TGEKP (SEQ ID NO: 45), GGRRGGGS (SEQ ID NO: : 46), or LRQRDGERP (SEQ ID NO: 47) (see, eg, Tang et al. (1996), J. Biol. Chem. 271, 15682-15686; Hennecke et al. (1998), Protein Eng. 11, 405-410) Or the residue may be any other amino acid that does not significantly reduce the ability to incorporate the variant without its adjacent added residues. Preferably, the number of contiguous amino acids other than the active peptide does not exceed 10 on either side of YGRKKRRQRRR (SEQ ID NO: 2). One suitable tat peptide containing an additional amino acid residue added adjacent to the C-terminus of YGRKKRRQRRR (SEQ ID NO: 2) is YGRKKRRQRRRPQ (SEQ ID NO: 48). However, preferably no amino acids are added adjacently.

Variants of the above-mentioned tat peptide with reduced ability to bind to N-type calcium channels are described in US patent application Ser. No. 60/904507, filed 03/02/2007. Such a variant comprises or consists of the amino acid sequence XGRKKRRQRRR (SEQ ID NO: 49), where X may be an amino acid other than Y or may be absent (if X is absent, G is a free N-terminal residue). Preferred tat peptides have the N-terminal Y residue replaced with F. Accordingly, a tat peptide containing or consisting of FGRKKRRQRRR (SEQ ID NO: 3) is preferred. Another preferred variant tat peptide consists of GRKKRRQRRR (SEQ ID NO: 1). Other tat peptides that promote uptake of pharmacological agents without inhibiting N-type calcium channels include those shown in Table 3.

  X can represent a free amino terminus, one or more amino acids, or a conjugated moiety.

  Internalized peptides can be coupled to pharmacological agents by conventional methods. For example, the agent can be linked to the internalization peptide via a chemical linkage (eg, via a binding or conjugating agent). A number of such drugs are commercially available and are reviewed by S.S. Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press (1991). Some examples of cross-linking reagents include J-succinimidyl 3- (2-pyridyldithio) propionic acid (SPDP) or N, N ′-(1,3-phenylene) bismaleimide; N, N′-ethylene- Bis (iodoacetamide) or other reagents with 6-11 carbon methylene bridges (relatively specific to sulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene (which are amino and tyrosine groups) And form an irreversible link). Other cross-linking reagents include p, p'-difluoro-m, m'-dinitrodiphenyl sulfone (which forms irreversible cross-linking with amino and phenolic groups); dimethyl adipimidate ( It is specific for amino groups); phenol-1,4-disulfonyl chloride (which mainly reacts with amino groups); hexamethylene diisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (which is mainly Including glutaraldehyde (which reacts with several different side chains) and disdiazobenzidine (which mainly reacts with tyrosine and histidine).

  In the case of a pharmacological agent that is a peptide, conjugation to the internalization peptide can be achieved by making a fusion protein comprising a peptide sequence, preferably fused to the internalization peptide at the N-terminus.

  Optionally, the pharmacological peptide fused to the tat peptide can be synthesized by solid phase synthesis or genetic recombination methods. Peptidomimetics can be synthesized using various processes and methodologies described in the scientific and patent literature. For example, Organic Syntheses Collective Volumes, Gilman et al., (Eds) John Wiley & Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9: 205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1: 114-119; Ostergaard (1997) Mol. Divers. 3: 17-27; Ostresh (1996) Methods Enzymol. 267: 220-234.

< IV. Inflammatory Response to Internalization Peptide > The present inventors have found that an internalization peptide (eg, tat) is capable of inducing an inflammatory response when administered to a subject. The inflammatory response is usually detected within 1, 5, 10, 20, 30, or 60 minutes of peptide administration but typically disappears within 24 hours of peptide administration (the peptide is not re-administered) Suppose). The inflammatory response is dose dependent. The inflammatory response typically recurs with similar intensity upon re-administration of the peptide.

  The inflammatory reaction is caused by mast cell degranulation, resulting in histamine and other inflammatory mediators (eg chemokines, cytokines, leukotrienes, lipases, proteases, kinins, cytokines, arachidonic acid derivatives (eg prostaglandins) ), Interleukin, and / or nitric oxide). Histamine and / or other released inflammatory mediators produce a number of inflammatory symptoms, including skin redness, fever, swelling, hypertension, and / or decreased pulse. Histamine release can cause vasodilation, bronchoconstriction, smooth muscle activation, endothelial cell separation (causing urticaria), pain, itching, increased capillary permeability, glandular hypersecretion, smooth muscle spasm, and Tissue infiltration of inflammatory cells (with gastric acid secretion and reduced release of neurotransmitters (eg, histamine, acetylcholine, norepinephrine, serotonin) can also occur.

< V. Anti-inflammatory Agents > A wide variety of anti-inflammatory agents are readily available to inhibit the types of inflammatory responses described above (see, eg, US Pat. No. 6,204,245, which is incorporated by reference). . One class of anti-inflammatory agents are antihistamine compounds. Such agents inhibit the binding of histamine to its receptor, thereby inhibiting the after-effects of inflammation that result from the above. Many antihistamines are commercially available, and some can be bought without a prescription from a pharmacy counter. Examples of antihistamines are azatadine, azelastine, valfroline, cetirizine, cyproheptadine, doxantrozole, etodroxizine, olin Hydroxyzine, ketotifen, oxatomide, pizotifen, proxicromil, N, N'-substituted piperazines, or terfenadine. Antihistamines vary in their ability to block antihistamines in the CNS as well as peripheral receptors by using second and third generation antihistamines that are selective for peripheral receptors. Acrivastine (Acrivastine), astemizole (Astemizole), cetirizine (Cetirizine), Loratadine (Loratadine), mizolastine (Mizolastine), levocetirizine (levocetirizine), desloratadine (Desloratadine), fexofenadine (Fexofenadine) the second and third generation This is an example of antihistamine. Antihistamines are widely used in oral and topical formulations.

  Another class of anti-inflammatory agents useful for inhibiting immune responses are corticosteroids. These compounds are transcriptional regulators and potent inhibitors of inflammatory symptoms resulting from the release of histamine and other compounds (resulting from mast cell degranulation). Examples of corticosteroids include cortisone (Cortisone), hydrocortisone (Cortef), prednisone (Deltasone), methicoten (Meticorten), orazone, prednisolone (Delta coatef, pediapred ( Pediapred, Prelone, Triamcinolone (Aristocort, Kenacort), Methylprednisolone (Medrol), Dexamethasone (Decadron, Dexone), Hexadolol Hexadrol)), and Betamethasone (Celestone). Corticosteroids are widely used in oral, intravenous and topical formulations.

  Another class of anti-inflammatory agents are mast cell degranulation inhibitors. This class of compounds includes 2-charboxylatochromon-5'-yl-2-hydroxypropane derivatives such as bis (acetoxymethyl) cromoglycate, disodium cromoglycate and nedocromil.

  Non-steroidal anti-inflammatory drugs (NSAIDs) can also be used. Such drugs include aspirin compounds (acetylsalicylic acids), non-aspirin salicylic acids, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, flurbiprofen, Includes ibuprofen, indomethacin, ketoprofen, meclofenamate, naproxen, naproxen sodium, phenylbutazone, sulindac, and tometin . However, the anti-inflammatory action of such drugs is less effective than that of antihistamines and corticosteroids.

  More powerful anti-inflammatory drugs (eg azathioprine, cyclophosphamide, leukeran, cyclosporine) can also be used, but they act slowly and / or It is not preferable because it relates to side effects. Biological anti-inflammatory agents (eg Tysabri® or Humira®) can also be used but are not preferred for the same reasons.

< VI. Conjugation > An alternative or additional way to suppress the inflammatory response induced by the internalizing peptide is to link the internalizing peptide to biotin or a similar molecule to form a conjugate. The conjugate retains the ability to promote cellular uptake of linked pharmacological agents, but induces a decrease in inflammatory response compared to the same internalizing peptide without biotin. Conjugated internalization peptides can be screened to confirm the desired uptake and resulting lack (reduction) of the immune response.

  Alternatives to biotin that can be used to form conjugates of internalization peptides include acetyl, benzoyl, alkyl groups (aliphatic), pyroglutamic acid, alkyl groups terminated by cycloalkyl groups, biotins with alkyl spacers , (5,6) -FAM included. Biotin or other molecules may be linked to the internalization peptide via amide chemistry, sulfamide chemistry, sulfone chemistry, and / or alkylation chemistry. Where and how to link biotin to tat. What are the characteristics of biotin suspected to contribute to reducing the inflammatory response?

< VII. Patients Accepted for Treatment / Prevention > As illustrated by the pharmacological agents listed in Table 1 and related conditions, a wide range of patients are amenable to treatment with the methods of the present invention. This method is particularly useful for patients who have a condition that would exacerbate any inflammation caused by the internalizing peptide (eg, patients suffering from hypertension, increased pulse, or other signs or symptoms of inflammation). is there. This method is also particularly useful for therapeutic methods that require high doses of pharmacological agents linked to internalizing peptides. Strictly speaking, it is the dose of the internalizing peptide rather than the linked pharmacological agent that determines the presence and extent of the inflammatory response, if any. However, the dose of internalization peptide is of course determined by the dose of linked pharmacological agent. For example, the inflammatory response may become evident at doses of internalization peptide greater than 1.5 mg / kg. For the treatment of some diseases, the effective amount of the pharmacological agent and the resulting linked internalization peptide is too low to induce an inflammatory response in most patients. Nevertheless, individual patients' sensitivity to inflammatory responses varies, and treatment with mild anti-inflammatory agents (eg, histamine) can still be a valuable preventive measure.

  One class of patients of particular interest is those who have or are at risk for a disease or condition characterized by excitotoxicity. Such diseases and conditions include stroke, epilepsy, hypoxia, trauma to the central nervous system not associated with stroke (eg, traumatic brain injury and spinal cord injury), Alzheimer's disease, and Parkinson's disease. The methods of the present invention are suitable for the treatment of both male and female patients having or at risk for these diseases and conditions.

Stroke is a condition resulting from blood flow disturbances in the central nervous system regardless of cause. Possible causes include embolism, bleeding, and thrombosis. Some neurons die immediately as a result of blood flow disturbances. These cells release molecules of their components, including glutamate, sequentially activate NMDA receptors, increase intracellular calcium levels, and increase intracellular enzyme levels to lead to further neuronal cell death (excitotoxicity cascade). The death of central nervous system tissue is called infarction. Infarct volume (ie, the volume of dead nerve cells resulting from a stroke) can be used as an indicator of the degree of pathological damage resulting from the stroke. The symptomatic effect depends on both the infarct volume and where it is located in the brain. The disability index can be used as a criterion for symptomatic disorders (eg, Rankin Stroke Prognostic Scale (Rankin, Scott Med J; 2: 200-15 (1957)) and the Barcel Index). The Rankine scale is based on direct assessment of the patient's overall condition as follows.

  The Basel Index is based on a series of questions about the patient's ability to perform the ten basic activities of daily life. The result is a score between 0 and 100, with lower scores indicating more severe disability (Mahoney et al., Maryland State Medical Journal 14: 56-61 (1965)).

  Alternatively, stroke severity / prognosis may be measured using the NIH stroke scale (available at world wide web ninds.nih.gov/doctors/NIH_Stroke_Scale_Booklet.pdf).

  The scale is based on the patient's ability to perform eleven groups of functions, including assessment of the level of patient awareness, motor function, sensory function, and language function.

  Ischemic stroke refers more specifically to a type of stroke caused by blockage of blood flow to the brain. The condition underlying this type of blockage is most commonly the occurrence of fat deposits in the inner layer of the vessel wall. This condition is called atherosclerosis. These fat deposits can cause two types of obstruction. Cerebral thrombosis refers to blood clots (thrombus and blood clot) that develop in clogged parts of blood vessels. “Cerebral embolism” generally refers to a thrombus that forms at another location in the circulatory system (usually the heart and upper thorax and cervical aorta). Some of the thrombus then loosens and breaks down, enters the bloodstream, and moves through the blood vessels in the brain until it reaches a blood vessel that is too small to pass. The second important cause of embolism is an irregular heartbeat known as arterial fibrillation. It creates a state where a thrombus can form in the heart, be removed, and move to the brain. Further possible causes of ischemic stroke are bleeding, thrombosis, arterial or venous dissociation, cardiac arrest, shock of any cause including bleeding, and iatrogenic causes (eg, leading to cerebrovascular or brain) Direct surgical damage to the blood vessels or cardiac surgery). Ischemic stroke accounts for approximately 83% of all stroke cases.

  Transient ischemic attacks (TIAs) are minor or warning strokes. In TIA, there are conditions that indicate ischemic stroke and typical warning signs of stroke occur. However, occlusion (thrombosis) occurs in a short time and tends to resolve itself via normal mechanisms. Patients who have undergone cardiac surgery have certain risks of transient cerebral ischemic attacks.

  Hemorrhagic stroke accounts for about 17% of stroke cases. It is due to weakened blood vessels that rupture and bleed into the surrounding brain. Blood accumulates and compresses surrounding brain tissue. Two common types of hemorrhagic stroke are intracerebral hemorrhage and subarachnoid hemorrhage. Hemorrhagic stroke results from a weakened vascular rupture. Possible causes of weakened vascular rupture include hypertensive bleeding (where high blood pressure causes vascular rupture), or another underlying cause of weakened blood vessels (eg, ruptured cerebrovascular malformations) (Eg, cerebral aneurysm, arteriovenous malformation (AVM), or spongy malformation)). Hemorrhagic stroke can also be caused by ischemic stroke, which weakens blood vessels at the infarct site, to hemorrhagic, or bleeding from primary or metastatic tumors in the central nervous system that contain abnormally weak blood vessels Can occur. Hemorrhagic stroke can also result from iatrogenic causes (eg, direct surgical damage to the cerebrovascular). An aneurysm is a weakened region of a blood vessel. If left untreated, the aneurysm continues to weaken until it ruptures and bleeds into the brain. An arteriovenous malformation (AVM) is a cluster of abnormally formed blood vessels. Spongiform malformations are venous abnormalities that can cause bleeding from weakened venous structures. Any one of these vessels can rupture and cause bleeding in the brain. Hemorrhagic stroke can also result from physical trauma. A hemorrhagic stroke that occurs in one part of the brain can lead to an ischemic stroke in another part through the lack of blood lost in the hemorrhagic stroke.

< VIII. Delivery of pharmacological agent together with anti-inflammatory agent> In a method in which a pharmacological agent linked to an internalizing peptide is administered together with an anti-inflammatory agent, the two entities are administered within a sufficiently close time. And the anti-inflammatory agent can inhibit an inflammatory response that can be induced by the internalizing peptide. The anti-inflammatory agent may be administered before, simultaneously with, or after the pharmacological agent, but is preferably administered before. The preferred time depends in part on the pharmacokinetics and pharmacokinetics of the anti-inflammatory agent. The anti-inflammatory agent is preferably administered at an interval prior to the pharmacological agent, so that the anti-inflammatory agent approaches a maximum serum concentration when the pharmacological agent is administered. Typically, the anti-inflammatory agent is administered between 6 hours before and 1 hour after administration of the pharmacological agent. Usually, the anti-inflammatory agent is present at a detectable serum concentration at several time points within the hour of pharmacological drug administration. The pharmacokinetics of many anti-inflammatory agents are widely known, and the relative timing of administration of anti-inflammatory agents can be adjusted accordingly. Anti-inflammatory agents are usually administered orally, intravenously, or topically depending on the appropriate drug. If the anti-inflammatory agent is administered at the same time as the pharmacological agent, the two may be administered as a combined formulation or separately.

  In a dosage regime consisting of an amount, frequency, and route effective to inhibit an inflammatory response to an internalizing peptide under conditions known to occur in the absence of an anti-inflammatory agent. Inflammatory agents are administered. An inflammatory response is inhibited when there is any reduction in signs or symptoms of inflammation as a result of anti-inflammatory agent administration. Symptoms of the inflammatory response include redness, fever, swelling, pain, tingling, itching, nausea, rash, dry mouth, numbness, and airway congestion. By measuring signs (eg, blood pressure or heart rate), the inflammatory response can be monitored. Alternatively, the inflammatory response may be assessed by measuring plasma concentrations of histamine or other compounds (released by mast cell degranulation). In practice, the dosage, dosing schedule, and route for administration of most of the anti-inflammatory agents described above are available in the "Physicians' Desk Reference" and / or from the manufacturer, and thus such anti-inflammatory agents. Can be used in the methods of the present invention consistent with such general guidance.

< IX. Treatment / Prevention Methods >< a) Treatment Methods > Administration effective to cure, reduce or inhibit further deterioration of at least one disease sign or symptom in a patient having the disease being treated A chimeric agent comprising a pharmacological agent attached to an internalizing peptide is administered in an amount, number, and route. A therapeutically effective amount is treated with a chimeric agent of the invention compared to an injury in a control group of patients (or animal models) suffering from a disease or condition not treated with the chimeric agent of the invention. In a group of patients suffering from a disease (or animal model), an amount of the chimeric agent significant enough to cure, reduce or inhibit further deterioration of at least one sign or symptom of the disease or condition being treated. means. An amount is also considered therapeutically effective if an individual treated patient achieves a more favorable prognosis than the mean prognosis of a control group of comparative patients not treated with the methods of the invention. A therapeutically effective regimen involves the administration of a therapeutically effective dose with the number and route of administration necessary to achieve the intended purpose.

  In the case of a patient suffering from a stroke or other ischemic condition, the chimeric agent in a dosage regime comprising an amount, frequency and route of administration effective to reduce the damaging effect of the stroke or other ischemic condition Is administered. If the condition requiring treatment is a stroke, its prognosis can be measured by infarct volume or disability index. If an individual treated patient has a disorder of 2 or less on the Rankine scale or a disorder of 75 or more on the Barcel scale, or the treated group has a significantly improved disability scale score distribution than the untreated comparison group Where indicated (ie, for lower disorders), the dose is considered therapeutically effective (see Lees et al., N Engl J Med 2006; 354: 588-600). A single dose of the chimeric drug is usually sufficient for the treatment of stroke.

< b) Prophylactic method > The present invention also provides a method and a composition for preventing a disease in a subject at risk of the disease. Usually such subjects exhibit an increased likelihood of developing a disorder (eg, condition, disease, disorder, or disease) compared to a control group. For example, the control group is randomly selected from a general group that has never been diagnosed with a disorder or has no family history (eg, combined from age, sex, race, and / or ethnicity) One or more individuals can be included. If it is found that a “risk factor” associated with the disorder is associated with the subject, the subject may be considered at risk for the disease. Risk factors include any activity, constitution, development, or nature associated with a given disorder, for example through statistical or epidemiological studies on a group of subjects. Thus, a subject may be classified as at risk for the disorder, even if the study identifying that underlying risk factor did not specifically include the subject. The frequency of transient cerebral ischemic attacks is increased in groups of subjects who have experienced heart surgery compared to groups of subjects who have not experienced heart surgery. Risk of cerebral ischemic attack.

  Other common risk factors for stroke include age, family history, gender, stroke, prior occurrence of transient ischemic or heart attack, hypertension, smoking, diabetes, carotid or other arterial disease, atrium Fibrillation, other heart diseases (eg, heart disease, heart failure, dilated cardiomyopathy, valvular heart disease, and / or congenital heart defect), high blood cholesterol, and diet (eg, saturated fat, trans fat, or High cholesterol diet).

  Internalize in patients who do not have the disease but are at risk for the disease, in an amount, frequency, and route sufficient to prevent, delay, or inhibit the occurrence of at least one sign or symptom of the disease A pharmacological agent linked to the peptide is administered. A prophylactically effective amount is a patient at risk of disease treated relatively with the drug compared to a control group of patients (or animal model) at risk of disease not treated with the chimeric drug of the present invention. In the group of (or animal models) means an amount of the chimeric agent significant enough to prevent, inhibit or delay at least one sign or symptom of the disease. An amount is also considered prophylactically effective if an individual treated patient achieves a more favorable prognosis than the mean prognosis of a control group of comparative patients not treated with the methods of the invention. A prophylactically effective regimen involves the administration of a prophylactically effective dose depending on the number and route of administration necessary to achieve the intended purpose. For the prevention of stroke in patients at imminent risk of stroke (eg, patients who have undergone cardiac surgery), a single dose of chimeric drug is usually sufficient.

< X. Pharmaceutical Composition, Dosage, and Route of Administration > The chimeric drug of the present invention can be administered in the form of a pharmaceutical composition. Pharmaceutical compositions are typically manufactured under GMP conditions. The pharmaceutical composition may be provided in unit dosage form (ie, a dose for a single administration). The pharmaceutical composition may be manufactured by the usual means of mixing, dissolving, granulating, dragee-forming, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. For example, a lyophilized chimeric agent of the invention may be used in the formulations and compositions described below.

  The pharmaceutical composition employs one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate the processing of the chimeric drug into a formulation that can be used medically. It can be formulated in a conventional manner. Proper formulation is dependent upon the route of administration chosen.

  Administration may be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Intravenous administration is preferred.

  Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic. For injection, the chimeric drug may be formulated in an aqueous solution, preferably a physiological phase such as Hanks's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the injection site). In a soluble buffer. The solution can contain formulations (eg, suspending, stabilizing, and / or dispersing agents).

  The chimeric agent, in another form, may be in powder form so that, prior to use, the composition is combined with a suitable medium (eg, sterile, pyrogen-free water).

  For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. This route of administration may be used to deliver the compound to the nasal cavity or for sublingual administration.

  For oral administration, the chimeric drug can be taken by a patient to be treated by a pharmaceutically acceptable carrier (eg, tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, and suspension). Etc.). For oral solid formulations (eg, powders, capsules and tablets), suitable excipients include fillers (eg, carbohydrates (eg, lactose, sucrose, mannitol and sorbitol)); cellulose formulations (eg, corn) Starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP)); granule; and binder. If desired, disintegrating agents (eg, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof (eg, sodium alginate)) may be added. If desired, solid dosage forms can be sugar-coated or enteric-coated using standard techniques. For example, for oral liquid formulations (eg, suspensions, elixirs, and solutions), suitable carriers, excipients, or diluents include water, glycols, oils, alcohols. Furthermore, you may add a fragrance | flavor, a preservative, a coloring agent, etc.

  In addition to the previously described formulations, the chimeric drug can also be formulated as a sustained release formulation. Such long lasting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compound may be a suitable polymer or hydrophobic material (eg, as an acceptable emulsion in oil) or an ion exchange resin, or a moderate water soluble derivative (eg, a moderate water soluble salt). ).

  Alternatively, other pharmaceutical delivery systems may be used. Liposomes and emulsions may be used to deliver the chimeric drug. Certain organic solvents (eg, dimethyl sulfoxide) may be used (but usually at the expense of greater toxicity). In addition, the compounds can be delivered using sustained release systems (eg, solid polymeric semipermeable matrices containing therapeutic agents).

Sustained release capsules can release the chimeric drug from weeks to over 100 days, depending on its chemical nature. Depending on the chemical nature and biological stability of the therapeutic agent, additional strategies for protein stabilization can be employed.

  Since the chimeric agents of the invention can include charged side chains or termini, they may be included in any of the above formulations as a free acid or base, or a pharmaceutically acceptable salt. Pharmaceutically acceptable salts are those salts that substantially retain the biological activity of the free base and are prepared by reaction with an inorganic acid. Pharmaceutical salts tend to dissolve more in aqueous solutions and other protic solvents than the corresponding free base form.

  A chimeric drug comprising an internalizing peptide linked to a pharmacological agent may be used at the same or lower dosage on a molar basis as that of the pharmacological agent alone, and is the same as that of the pharmacological agent alone And the same pharmacological agent alone may be administered to treat the same disease.

  For the treatment of stroke, a preferred dose range includes 0.001 to 20 μmol chimeric peptide or peptidomimetic per kg patient body weight, and optionally includes 0.03 to 3 μmol chimeric peptide per kg patient body weight. In some methods, 0.1 to 20 μmol chimeric peptide or peptidomimetic is administered per kg patient body weight. In some methods, 0.1-10 μmol chimeric peptide or peptidomimetic per kg patient body weight, more preferably about 0.3 μmol chimeric peptide per kg patient body weight. In another embodiment, the dose range is 0.005 to 0.5 μmol of chimeric peptide or peptidomimetic per kg patient body weight. The dose per kg body weight can be converted from rat to human by dividing by 6.2 to correct for different surface area to mass ratios. The dosage can be converted from moles to grams by multiplying by the molecular weight of the chimeric peptide or peptidomimetic. Suitable dosages of chimeric peptides or peptidomimetics for use in humans include 0.001 to 5 mg / kg patient body weight, or more preferably 0.005 to 1 mg / kg patient body weight, or 0.05 to 1 mg / kg, or 0.09 Contains 0.9mg / kg. For absolute weights for a 75 kg patient, these doses are calculated as 0.075 to 375 mg, 0.375 to 75 mg, or 3.75 mg to 75 mg, or 6.7 to 67 mg. For example, when rounded to include changes in the patient's body weight, the dosage is usually within 0.05 to 500 mg, preferably 0.1 to 100 mg, 0.5 to 50 mg, or 1 to 20 mg.

  The dosage of the chimeric agent depends on the subject being treated and depends on the subject's weight, the severity of the affliction, the method of administration, and the judgment of the prescribing physician. The therapy may be repeated intermittently while symptoms are detectable or even when they are not detectable. Treatment may be provided alone or in combination with other drugs. For the treatment of stroke, dosing of the pharmacological agent linked to the internalizing peptide is usually administered within 24 hours from the onset of stroke, preferably within 6 hours from the onset of stroke.

< XI. Kit > A kit is provided for carrying out the method of the present invention. The kit includes one or more pharmacological agents of interest coupled to an internalizing peptide. The internalization peptide may be biotinylated and / or the kit may contain an anti-inflammatory agent. The instant kit optionally includes a means for administering a pharmacological agent and / or an anti-inflammatory agent. The kit can also include one or more buffers, additives, fillers, or diluents. The kit may provide one or more printed instructions regarding administration and subsequent dosage regimens.

< XII. Screening Method ><A . Measurement of Internalization Activity > Mutants (mutants of tat or other internalization peptides) can be tested for permeability activity in animals. Internalization peptides may be tested alone or when linked to an active agent (eg, an active peptide such as KLSSIESDV (SEQ ID NO: 5)). The internalization peptide (optionally but linked to the active agent (eg, peptide)) is preferably labeled with a fluorescent label (eg, dansyl chloride). The internalizing peptide is then injected into the periphery of the animal (eg, mouse). For example, intraperitoneal or intravenous injection is suitable. Approximately 1 hour after injection, mice are sacrificed and perfused with fixative (3% paraformaldehyde, 0.25% glutaraldehyde, 10% sucrose, 10 U / mL heparin in saline). The brain is then removed, frozen, and sectioned. Sections are analyzed for fluorescence using a confocal microscope. Optionally, relative to positive and negative controls, internalization activity is measured from fluorescence. A suitable positive control is a standard tat peptide linked to the same active peptide (if present) as the internalization peptide to be tested. A suitable negative control is a fluorescently labeled active peptide that is not linked to an internalization peptide. Unlabeled media can also be used as a negative control.

  Similar experiments can be performed to test mutants (mutants of tat or other internalizing peptides) in cell culture (see US Patent Publication No. 20030050243). A fluorescently labeled mutant tat peptide (optionally linked to the active peptide) is added to the cortical neuron culture. Uptake is measured using a fluorescence microscope for several minutes after addition. Increased uptake relative to positive and negative controls can be measured as described for experiments on uptake in animals.

< 2. Measurement of activity in the treated stroke >
The activity of a chimeric drug comprising an internalizing peptide linked to the drug can be tested in various animal models of stroke. In one such model, adult male Sprague Dawley rats are exposed to transient middle cerebral artery occlusion (MCAO) by intraluminal sutures (36, 37) for 90 minutes. Animals are fasted overnight and injected with atropine sulfate (0.5 mg / kg IP). After 10 minutes, anesthesia is performed. Rats are intubated by mouth, mechanically ventilated, and anesthetized with pancuronium bromide (0.6 mg / kg IV). Body temperature is maintained at 36.5-37.5 ° C. using a heating lamp. Blood pressure is continuously measured using polyethylene catheters in the femoral artery and vein, and blood is collected for gas and pH measurements. Transient MCAO effectively occludes the middle cerebral artery by introducing poly-L-lysine-coated 3-0 monofilament nylon thread (Harvard Apparatus) into the Willis artery ring via the internal carotid artery for 90 minutes It is realized by doing. This creates an extensive infarct that surrounds the cerebral cortex and basal ganglia. Animals are treated with either the chimeric drug to be tested or either a negative or positive control. Treatment may be either before inducing ischemia or up to 1 hour later. The negative control can be a medium. The positive control can be the tat-NR2B9c peptide (YGRKKRRQRRRKLSSIESDV (SEQ ID NO: 6)) previously shown to be effective. Chimeric drug is delivered by single intravenous bolus administration (3 nmoles / g) 45 minutes before MCAO. After administering the compound to the animal, the infarct volume and / or disability index is measured. Infarct volume is usually measured 24 hours after treatment. However, it may be measured at a later time (eg, 3, 7, 14, or 60 days). The disability index may be monitored, for example, at 2 hours after treatment, 24 hours after treatment, 1 week and 1 month after treatment. Chimeric agents that show a statistically significant reduction in infarct volume and / or disability index compared to control animals not treated with the compound are identified as having useful activity in the practice of the methods of the invention. The

  Similar experiments can be performed in animals exposed to permanent ischemia. As described by Forder et al., Permanent ischemia of the middle cerebral artery pial vascular can be performed (Am J Physiol Heart Circ Physiol 288: H1989-H1996 (2005)). Briefly, a catheter is inserted into the right ECA using a PE10 polyethylene tube. A 6-8 mm cranial window is made in the right somatosensory cortex (bregma 2 mm caudal and 5 mm lateral) with the skull exposed through a midline incision. Visualize the pial arteries by injecting a small bolus (10-20 μL) of the biological dye patent blue violet (10 mMol / L; Sigma) dissolved in normal saline into the ECA. The same three pial artery MCA branches are electrocauterized and the dye injection is repeated to ensure interruption of blood flow through the cautery artery. The incision is then closed and the animal is returned to the cage and free access to food and water. This model of permanent ischemia produces a small, highly reproducible infarct limited to the cortex based on the coagulated terminal pial artery.

  The left middle cerebral artery can be occluded by the intraluminal suture method described by Longa (Stroke 20, 84-91 (1989)). Briefly, the left common carotid artery (CCA) is exposed through a midline cervical incision. And it dissects from the bifurcation part to the skull base, without including the surrounding nerves and the membrane. The occipital artery branch of the external carotid artery (ECA) is then isolated, and these branches are dissected and coagulated. The ECA is dissected further and then coagulated with the terminal tongue and maxillary artery branch separated. The internal carotid artery (ICA) is isolated, separated from the adjacent vagus nerve, and the wing palate artery is ligated near its origin. A 4 cm long tip of 3-0 monofilament nylon suture (Harvard Apparatus) is fired and rolled to achieve a tip diameter of 0.33 to 0.36 mm and a tip length of 0.5 to 0.6 mm, and Coated with poly-L-lysine (Belayev et al., 1996). Sutures are introduced through the CCA and advanced into the ICA and from there into the Willis annulus (approximately 18-20 mm from the carotid bifurcation), effectively occluding the middle cerebral artery. Around the intraluminal nylon suture, tighten the silk suture around the CCA to tighten it and permanently occlude the middle cerebral artery.

  <Example 1> <Impact of gender difference on neuroprotective efficacy of Tat-NR2B9c> The neuroprotective efficacy of Tat-NR2B9c was compared with the in vivo buffy coat 3 vascular occlusion (P3VO) model (Forder JP, Munzenmaier DH, Greene AS, rat Angiotensin II type 1 receptor blockade with angiogenesis protection from local ischemia, Am J Physiol Heart Circ Physiol 2005 April; 288 (4): H1989-H1996) was evaluated in both male and female rats.

< Method ><Animal> Adult Sprague-Dawley rats (10-12 weeks old) (male ~ 300g, male) before performing permanent puffal vascular occlusion (P3VO) on the 3 final branches of middle cerebral artery on whisker barrel cortex Females-25 Og) were fasted for 12-18 hours. Tat-NR2B9c was tested in male rats with the addition of a saline control group (each group, n = 8). Tat-NR2b9c and saline control were tested in female rats (each group, n = 8). The researchers blinded the treatment group from the time of surgery to the analysis of infarct size.

  <General procedure> Rats received 0.5 ml / kg intramuscular injection (ketamine (100 mg / kg), acepromazine (2 mg / kg) and xylazine (5 mg / kg) one third of the initial dose required. And anesthetized. The anal temperature probe was inserted and the animal was placed on a heating pad maintained at 37 ° C. A catheter was inserted into the right external carotid artery (ECA) using PE10 polyethylene tubing for dye injection. The skull was stripped through a midline incision and the surface was scraped to remove tissue, and the temporal muscle was disconnected from the right skull. Using a dissecting microscope and a pneumatic dental drill, the right somatosensory cortex (bregma to caudal 2 mm and caudal side by drilling the skull into a quadrilateral and lifting that fragment of the skull while keeping the dura intact. A 6x4mm cranial window was prepared on the side (5mm). After rinsing with artificial cerebrospinal fluid, a small bolus (10 mmol / L; Sigma) of a biological dye dissolved in normal saline to demonstrate passage through blood vessels on the surface of the cortex 10-20 μL) was injected into the right external carotid artery. Three important arterial branches of MCA surrounding the barrel cortex were selected and electrocauterized through the dura mater. After ablation, bolus injection and dye passage were repeated to ensure that passage through the ablated artery was blocked. The quadrilateral of the skull was placed over the window and the skull was sutured. The catheter was removed from the ECA, the ECA was ligated, and the anterior neck was sutured. One hour after the onset of local occlusion, 0.3 ml of drug (3 nMol / g body weight) or saline control was infused through the tail vein at a rate of 0.06 ml / min. Each rat was returned to an independent cage under a heating lamp to maintain body temperature until the rat was fully recovered. Feed and water were supplied freely.

  <Collecting brain tissue and analyzing infarct size> After 24 hours of surgery, the animals were re-anesthetized with 1 mL of pentobarbital, and the brain was rapidly collected. Coronal sections were removed from the infarct area and incubated in 2% triphenyltetrazolium chloride (TTC) for 15 minutes at 37 ° C. Images were captured with a scanner and brain sections were stored at -80 ° C. Infarct size was measured as a percentage of each rat's hemisphere in this study. After measuring infarct size, the animals were divided into groups. Comparisons were made as mean ± SE between treatment groups.

  Results and Conclusions The P3VO model of stroke in rats produced strong and reproducible infarcts in both male and female SD rats. The tat-NR2B9c peptide is neuroprotective in both males and females since significantly reduced infarct size is observed 24 hours after P3VO surgery (Figure 1). Treatment with Tat-NR2B9c (3 nM / g) 1 hour after the stroke dramatically reduced infarction in bisexual animals (Figure 1). This neuroprotective response appeared more pronounced in females than in males, as a complete lack of infarction was seen in female rats treated with equivalent concentrations of Tat-NR2B9c. However, the control treated with saline shows that the mean infarct size is smaller (71%) in female rats than in male rats.

<Example 2><Peptide containing Tat sequence induces mast cell degranulation with histamine release in vitro>< Method ><Cellculture> C57 mice are sterilized with 70% ethanol and skin and connective tissue are removed The femur was dissociated. Bone marrow cells are harvested and reconstituted in OPTI-MEM (Gibco) (containing 5% heat-inactivated FBS, 6% WEHI conditioned medium (as a source of IL-3), and 55 μM □ -2 mercaptoethanol) Suspended. The cells were cultured at about 1 × 10 ⁶ cells / mL. Two days later, the cells were harvested and centrifuged, and the pellet was plated on a new plate using fresh medium. Fresh WEHI conditioned medium was added each week. The cells were cultured for about 4 weeks after they became> 95% mast cells and used for mast cell degranulation assays.

<Mast cell degranulation assay> Tryptase activity was measured using a mast cell degranulation assay kit (CHEMICON, Temecula, CA). After isolation, the cells were washed and resuspended at about 1 × 10 ⁶ cells / mL in 1 × assay buffer. For treatment with TAT-NR2B9C or other peptides, 50 μL of the following concentration solution (0.125 mg / mL, 1.25 mg / mL, 12.5 mg / mL, or 125 mg / mL) was added to the cell suspension: . 500 nM A23187 (Calcimycin), an inducer of tryptase release in mast cells, was used as a positive control. Cells were incubated for 60 minutes at 37 ° C., 5% CO ₂ . The cell suspension was centrifuged at 700xg and the supernatant was carefully collected. The assay mixture (provided in the kit) was prepared in a 96-well microtiter plate. The colorimetric reaction was initiated by adding 20 μL tryptase substrate to each experiment and control well. Samples were incubated for 60 minutes at 37 ° C. Absorbance was measured at 405 nm in a microplate radar.

The following treatment was used to induce mast cell degranulation.
1) Negative control (assay buffer without any peptide)
2) Positive control (calcium ionophore A23187)
3) Tat-NR2B9C
4) Tat-derived sequence of T-NR2B9C without PSD-95 binding sequence
5) NR2B9C without Tat sequence but with PSD-95 binding sequence of TAT-NR2B9C
6) AA (20 amino acid peptide containing a Tat sequence fused to the 9 carboxy terminal amino acids of the NMDA NR2B subunit, but with a 2 amino acid mutation that prevents it from binding to PSD-95)

  Approximate the maximum serum concentration achieved in dogs receiving a 10 mg / kg dose of Tat-NR2B9C in several animal experiments described below (based on assuming blood volume to be 8% of total body weight) Therefore, all peptides were added at a concentration of 125 mg / mL.

  <Results and Conclusion> As can be seen in FIG. 2, all the peptides containing the Tat transduction domain caused mast cell degranulation. However, the NR2B9c peptide (without the Tat sequence) was not. In vitro mast cell degranulation assays were performed in the absence of antibody. Thus, any mast cell degranulation cannot be due to an immune phenomenon. In particular, using RT-PCR and Western blot, we examined whether mast cells contained PSD-95, a therapeutic target for Tat-NR2B9c. We could not detect PSD-95 in these cells (results not shown). This result provided further evidence that mast cell degranulation did not appear to be caused by the interaction between TAT-NR2B9c and PSD-95.

  In further experiments, we determined that the extent of mast cell degranulation by Tat-NR2B9c and that by AA peptide are dose dependent. Specifically, when the concentration of Tat-NR2B9c is further increased, an increase in mast cell degranulation is induced as shown in FIG.

In further experiments, we investigated the effect of Tat-NR2B9c sequence variation on mast cell degranulation. The following compounds (all at 50 uM) were tested using the same assay.

  As can be seen in FIG. 4, all compounds containing Tat sequences and Tat peptide sequences induced mast cell degranulation. However, NR2B9c alone did not elicit this response.

  Example 3 <Conjugation of peptide containing Tat sequence cannot induce mast cell degranulation in vitro> Using the method described in Example 2, certain modifications to Tat-containing peptides (eg: The effect of conjugation on mast cell degranulation was examined. Modified peptides include Tat-NR12B9c, D-isomer of Tat-NR2B9c (named D-Tat-NR2B9c), biotin-conjugated Tat-NR2B9c, biotin-conjugated AMP-KLSSIESDV (SEQ ID NO: 5). As shown in FIG. 5, biotin-conjugated Tat or AMP peptide was unable to induce mast cell degranulation.

  <Example 4> <Tat-NR2B9c induces increased histamine levels and histamine response in animal studies with beagle dogs> Untreated 14-day toxicity study by intravenous administration of Good Laboratory Practice (GLP) Beagle dogs (3 / sex / group) (CRM study number: 501448) were used. Among them, animals were injected daily with 0, 0.25, 1.0, or 10 mg / kg Tat-NR2B9c. Blood samples (approximately 1 mL) were taken from all animals 1, 6, and 12 days before dosing and 5 and 15 minutes after injection. Blood samples were collected into tubes containing EDTA by venipuncture (jugular vein, saphenous vein, and cephalic vein). Samples were then centrifuged (within 30 minutes of collection) at 2700 rpm for 10 minutes in a chilled centrifuge (approximately 4 ° C.). Plasma was separated into appropriately labeled second tubes and stored at −80 ° C. until CRM analysis. Plasma samples were used to examine histamine levels. Samples from animals that received Tat-NR2B9c intravenously were analyzed using a justified method.

All animals receiving 10 mg / kg Tat-NR2B9c had treatment-related clinical signs (nose mouth, gingiva (also described as pale), auricle, periorbital area, and limb redness And is often associated with swelling. These effects were associated with lethargy and non-tactile pulses, and the attending veterinarian characterized it as a severe hypotensive response. These effects were observed daily starting from the first day of dosing and continuing through the 14 day dosing period, but there was no apparent adaptation to the animals. These effects were not due to the development of antibody-based immune responses. This is because these animals were not exposed to Tat-NR2B9c until the first day of dosing and no increased severity of the response was observed in 14 days of treatment. Specifically, increased histamine levels were observed immediately following the first dose of Tat-NR2B9c to these untreated bagel dogs (see Table 6 for a summary of dog plasma histamine levels) ). These animals have never been exposed to Tat-NR2B9c and therefore should not have memory T cells or circulating antibodies to Tat-NR2B9c. Also, during the 14-day repeated dose toxicity study at any dose level, no consistent increase in histamine levels was observed, indicating no expansion of antigen-specific responses. Thus, the observed increase in histamine levels by Tat-NR2B9c is a result of direct mast cell degranulation rather than an antigen-specific antibody response.

  <Cardiovascular effects of Tat-NR2B9c showing histamine release in dogs> In the GLP cardiovascular telemetry test (CRM study number: 691106) in unrestrained awake beagle dogs, there was a 3 day drug holiday between dose levels Thus, 6 animals (3 males and 3 females) were administered with increasing doses of Tat-NR2B9c (0.25, 1.0, or 5.0 mg / kg peptide total). At 0.25 or 1.0 mg / kg, no effect on blood pressure was observed. At 5 mg / kg, a transient drop in blood pressure was observed in 4 out of 6 dogs, lasting about 30 minutes. The finding that the drop in blood pressure may be dose related indicates that the drop was not due to an allergic (antibody-mediated) immune response. Furthermore, the time frame between the low dose (0.25 mg / kg) and the high dose (5.0 mg / kg) is about 6 days. That is, a period that is insufficient to allow an immune response to be generated. The action is therefore caused by the direct degranulation of mast cells causing histamine release.

  Additional GLP circulatory system remote in unrestrained awake beagle dogs to obtain detailed circulatory system information at the highest dose level tested in a 14-day canine repeated dose toxicity study (ie, 10 mg / kg) A measurement test was conducted (CRM test number: 691429). Six animals (3 males, 3 females) received vehicle in the morning and 10 mg / kg Tat-NR2B9c (at least 4 hours between doses) in the afternoon of the same day. An increase in heart rate was observed in the treated animals up to 15 minutes after dosing, with a maximum effect observed at 10 minutes in males and females. From 5 to 10 minutes after dosing, decreased blood pressure values (up to 62%) were observed in individual animals. The female animals used in this additional circulatory system test were treated animals that were obtained from the Charles River colony and were previously used in the first circulatory system test of Tat-NR2B9c (CRM test number: 691106). there were. The effect observed at 10 mg / kg in CRM Trial No. 691429 corresponds to the clinical signs observed in the 14-day canine toxicity study (CRM Trial No. 501448) and was tested in CRM Trial No. 691106 It had a more severe blood pressure effect observed in treated animals than was observed at the highest dose level (5 mg / kg). These results again point to direct degranulation of mast cells.

  We performed a non-GLP study of the effect of Tat-NR2B9c on blood pressure in anesthetized rats receiving 50 mg / kg Tat-NR2B9c. This dose was also chosen for rats because this dose reduces tidal volume, respiratory rate, and minute ventilation derived therefrom. In one experiment, 5 Sprague-Dawley rats were given a 50 mg / kg Tat-NR2B9c bolus dose for 3 minutes. Blood pressure was monitored via a femoral artery catheter.

  A transient decrease in mean arterial pressure occurred in all animals as shown in FIG. Another experiment in which six animals were similarly tested showed similar results. As discussed above in the case of dogs, these responses in rats were also observed in untreated animals that did not have any prior opportunity to generate an immune response against Tat-NR2B9c. These data provide evidence in the second species of mast cell degranulation by peptides containing the Tat sequence.

  <Inflammatory Response Showing Histamine Release in Dogs> A non-GLP study was conducted to determine the dose range of Tat-NR2B9c administered to Beagle dogs by single, slow intravenous injection. Two animals (one male beagle and one female) were intravenously dosed with Tat-NR2B9c seven times. There was a 3-4 day drug holiday between the doses. The first dose was given at 2.5 mg / kg. Since the animal did not show any signs of toxicity, the second dose was administered at 7.5 mg / kg. Male animals exhibited angioedema of the head soft tissue and urticaria-type reaction, especially on the ventral side of the abdomen. There was no response in female dogs. Vital signs (heart rate, blood pressure, respiratory rate, and body temperature) remained within normal physiological ranges in both animals. The third dose was given at 12.5 mg / kg. After dosing, angioedema and urticaria were observed in both animals. The response in male dogs was moderate, and in female animals the response was mild. The next dose was set at 20.0 mg / kg. After dosing, male animals fell into shock and blood pressure (BP) and pulse were not detected. The animals were treated with i.v. administration of benedryl and dexamethasone. The BP 5 minutes after dosing was recorded as 37/13 mm Hg (normal BP in dogs ~ 160/90). It was decided not to dose female animals.

  In order to better understand the type of response seen with the previous dosing, the following dosing was given. The fifth and sixth doses were set at 2.5 and 5.0 mg / kg. At 2.5 mg / kg, no other side effects were observed in any animal except for redness of the ears and face of male dogs. At 5.0 mg / kg, there was a moderate response in male animals but no response in female dogs.

  It was concluded that high dose Tat-NR2B9c can induce marked transient hypotension and urticaria-like skin reaction. These responses appeared to be dose dependent, with male animals appearing to be more sensitive to the test article than female animals.

  <Example 5> <Treatment with antihistamines prevents symptoms induced by Tat-NR2B9c in dog> After pre-administration with 1 mg / kg Benedryl administered 30 minutes before Tat-NR2B9c Both animals of Example 4 were administered 12.5 mg / kg Tat-NR2B9c. There was slight redness of the ear dermis in male animals. Male animals also vomited 15-20 minutes after administration of Tat-NR2B9c. No response was observed in female dogs. Thus, pre-administration of the antihistamine drug Benedryl blocked the vascular neuroedema and urticaria responses previously observed with the same dose level of Tat-NR2B9c in both animals. This result shows that antihistamines (eg, benedryl) and corticosteroids (eg, dexamethasone) effectively treat the adverse events of mast cell degranulation.

  Taken together, these results indicate that administration of Tat-NR2B9c induced an increase in blood histamine levels in experimental animals, the increased histamine levels were due to mast cell degranulation, and this response was anti-histamine Direct experimental evidence that treatment with drugs and corticosteroids may constitute an effective means to administer other compounds containing Tat-NR2B9c and protein transduction domains (eg Tat) provide.

<Example 6><Direct evidence that Tat-NR2B9c induces an increase in blood histamine in human>< Method > We conducted safety, tolerability, and pharmacokinetic studies of Tat-NR2B9c in humans. Subjects were either normal, healthy, non-smoking males 18 years of age or older, or postmenopausal or surgically sterile female subjects. Subjects receive Tat-NR2B9c (Lot No: 124-134-001B) by intravenous infusion (10 ± 1 min) or placebo (Phosphate Buffered Saline) (Lot No: 124 -134-001A). Four subjects were dosed in each of cohorts 1 to 3, and 10 subjects were dosed in each of cohorts 4 to 8. All 62 subjects completed the study. The treatment period for each cohort is as follows: Cohort 1: September 14, 2006, Cohort 2: September 26, 2006, Cohort 3: October 6, 2006, Cohort 4: October 20, 2006, Cohort 5: November 6, 2006, Cohort 6: December 4, 2006, Cohort 7: December 17, 2006, Cohort 8: February 25, 2007.

  <Blood collection time points> During the study period, 11 blood samples were collected from each subject at the following time points for pharmacokinetic analysis: 0.00 (pre-dose), 0.08 (5 minutes), 0.17 to 0.25 (after administration) Exactly 10-15 minutes from the end of each independent drug infusion), 0.33 (20 minutes), 0.50, 0.75, 1.00, 2.00, 6.00, 12.00, and 24.00 hours. In addition, eight blood samples were collected from each subject at the following time points for histamine analysis: 0.00 (pre-dose), 0.08 (5 minutes), 0.17 (10 minutes), 0.25, 0.50, 1.00 after dosing. , 2.00, and 24.00 hours.

  <Safety assessment> A safety study was conducted in all subjects who received at least one dose during the course of the study. The incidence of all adverse events (AEs) was counted by treatment and number of subjects. Vital sign absolute values, electrocardiogram (ECG) parameters, experimental parameters and physical examination were also documented and marked out of normal range. The shift from the baseline value was tabulated. AEs were documented using research terminology and the Medical Dictionary for Regulatory Activities (MedDRA) terminology.

< Results ><Part 1: Effect of Tat-NR2B9c on blood histamine levels> A summary of abnormal histamine results from dosing is described in Table 7. Seven of 8 subjects in the 3.75 mg / kg dose group had histamine levels higher than 10 nmol / L (mean 24.3 nmol / L; maximum 39.8 nmol / L) 10 minutes after starting NA1 administration. Three of the subjects still had histamine levels higher than 10 nmol / L (mean 15.3 nmol / L; maximum 20.3 nmol / L) 15 minutes after the start of NA1 administration.

Other than the 3.75 mg / kg dose group, no treatment group had significantly abnormal levels of histamine. The placebo and 0.375 mg / kg dose groups each had one subject with elevated histamine levels at one time point, but these results were at screening and 2.00 hours after dosing, respectively. there were. All abnormal histamine results returned to normal within 24.00 hours of drug administration.

  <Part 11: Safety data> Forty subjects who participated in the study experienced a total of 168 adverse events (AEs) during the study. Most AEs were mild in severity. Six of 16 subjects who received placebo treatment (37.5%) experienced at least one AE, while 34 of 46 subjects who received active treatment (73.9%) had at least one Experienced AE. Subjects in the 2.60 and 3.75 mg / kg dose groups experienced significantly more AEs than subjects in the lower dose group. No serious adverse events (SAEs) were reported. The most common adverse events experienced by subjects receiving Tat-NR2B9c were heat sensation (13/46; 28.3%), pruritus (12/46; 26.1%), flushing (10/46) 21.7%), and thirst (9/46; 19.6%). All AEs resolved with the exception of 2 cases of increased blood glucose, and the subject could not be followed up.

  The incidence of AEs in the 2.60 and 3.75 mg / kg dose groups was higher than the incidence of AEs in the placebo, 0.02, 0.08, 0.20, 0.375, 0.75, and 1.50 mg / kg dose groups. Several AEs were frequently reported at doses of Tat-NR2B9c ≧ 2.60 mg / kg. These include (1) decreased blood pressure, (2) tingling (abnormal sensory perception), (3) numbness (hyposensory), (4) redness (erythema), (5) rash, (6) itching (so Mania), (7) dry mouth, (8) nausea, (9) heat sensation, and (10) flushing. The onset of these adverse events occurred concurrently with study drug administration and was probably related to study drug.

  In preclinical studies with Tat-NR2B9c, elevated histamine levels were observed in the high dose group and could be the cause of side effects (including swelling, redness, and hypotension). In the current study, histamine levels increased in 7 of 8 subjects at the highest dose group (3.75 mg / kg) 10 minutes after the start of intravenous drug administration and 15 minutes after drug administration Later, 3 of these subjects remained elevated, after which the level returned to the normal range. Most of the AEs in the 3.75 mg / kg dose group were observed during the same time frame in which histamine levels were elevated. This was because elevated histamine levels were the cause of the most frequently reported AEs (decreased blood pressure, tingling, numbness, redness, rash, itching, dry mouth, nausea, warmth, and flushing) To suggest.

Most of the adverse events listed were also observed in animal preclinical studies, where the maximum tolerated dose (MTD) was established at 12.5 and 100 mg / kg in dogs and rats, respectively. Most of the AEs in the 2.60 and 3.75 mg / kg dose groups were not observed, or were observed in only one subject in the dose group between 0.02 and 1.50 mg / kg. This suggests that AEs observed at higher doses of Tat-NR2B9c were minimal or absent at lower dose ranges.

  Although the invention has been described in detail for purposes of clarity of understanding, it will be apparent that certain modifications may be practiced within the scope of the appended claims. All publications and patent documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each was individually indicated. Unless apparent from the context, any element, embodiment, step, function, or aspect of the invention can be performed in combination with any other.

Claims (28)

A method of delivering a pharmacological agent to a subject comprising:
Administering to the subject the pharmacological agent linked to an internalizing peptide;
Administering an anti-inflammatory agent to the subject,
The method wherein the anti-inflammatory agent inhibits an inflammatory response induced by the internalizing peptide.   2. The method of claim 1, wherein the anti-inflammatory agent is antihistamine.   2. The method of claim 1, wherein the anti-inflammatory agent is a corticosteroid.   2. The method of claim 1, wherein the internalization peptide is a tat peptide.   5. The method of claim 4, wherein the tat peptide has an amino acid sequence comprising RKKRRQRRR (SEQ ID NO: 51) or GRKKRRQRRR (SEQ ID NO: 1).   5. The method of claim 4, wherein the tat peptide has an amino acid sequence comprising YGRKKRRQRRR (SEQ ID NO: 2).   5. The method of claim 4, wherein the tat peptide has an amino acid sequence comprising FGRKKRRQRRR (SEQ ID NO: 3).   5. The method of claim 4, wherein the tat peptide has an amino acid sequence comprising GRKKRRQRRRP (SEQ ID NO: 4).   2. The method of claim 1, wherein the pharmacological agent is a peptide.   2. The method of claim 1, wherein the pharmacological agent is KLSSIESDV (SEQ ID NO: 5).   Use of an anti-inflammatory agent in the manufacture of a medicament for inhibiting an inflammatory response induced by an internalizing peptide linked to a pharmacological agent.   A kit comprising a pharmacological agent linked to an internalization peptide, and an anti-inflammatory agent that inhibits an inflammatory response induced by the internalization peptide.   An internalization peptide linked to biotin that has a reduced ability to induce an inflammatory response compared to an internalization peptide without biotin.   A method of delivering a pharmacological agent to a subject, the method comprising administering to the subject the pharmacological agent linked to an internalization peptide, wherein the internalization peptide is biotinylated. And the biotinylation reduces the ability of the internalizing peptide to induce an inflammatory response compared to the internalizing peptide without the biotin. A method of influencing the treatment or prevention of diseases mediated by excitotoxicity, comprising:
In a dosage regime effective to effect efficacy in the treatment or prevention of the disease, a pharmacological agent that inhibits the binding between PSD95 and NDMAR 2B is linked to an internalizing peptide, or has a risk of having the disease To a subject with
Administering an anti-inflammatory agent to the subject, whereby the anti-inflammatory agent inhibits an inflammatory response induced by the internalizing peptide.   16. The method of claim 15, wherein the pharmacological agent is an NMDAR receptor PL peptide. 16. The method of claim 15, wherein the internalization peptide is a tat peptide.   An amino acid sequence wherein the internalizing peptide comprises RKKRRQRRR (SEQ ID NO: 51), GRKKRRQRRR (SEQ ID NO: 1), YGRKKRRQRRR (SEQ ID NO: 2), FGRKKRRQRRR (SEQ ID NO: 3), or GRKKRRQRRRPQ (SEQ ID NO: 4) 16. The method according to claim 15, comprising:   16. The method of claim 15, wherein the subject is a female.   16. The method of claim 15, wherein the disease is stroke.   16. The method of claim 15, wherein the subject is at risk for a transient cerebral ischemic attack as a result of undergoing cardiac surgery. A method of influencing the treatment or prevention of diseases mediated by excitotoxicity, comprising:
In a dosage regime effective to effect efficacy in the treatment or prevention of the disease, a pharmacological agent that inhibits the binding between PSD95 and NDMAR 2B is linked to an internalizing peptide, or has a risk of having the disease Administering to a subject with
The method wherein the internalization peptide is biotinylated and the biotinylation reduces the ability of the internalization peptide to induce an inflammatory response. A method of influencing the treatment or prevention of diseases mediated by excitotoxicity, comprising:
In a dosage regime effective to effect efficacy in the treatment or prevention of the disease, a pharmacological agent that inhibits the binding between PSD95 and NDMAR 2B is linked to an internalizing peptide, or has a risk of having the disease Administering to a female subject.   24. The method of claim 23, wherein the internalization peptide is a tat peptide.   In a method for delivering a pharmacological agent linked to an internalization peptide to a subject, the improvement is an immunosuppression wherein the internalization peptide is biotinylated or inhibits an inflammatory response induced by the internalization peptide A method that is either administered with an agent.   26. The method of claim 25, wherein the internalization peptide is a tat peptide. A method of inhibiting an inflammatory response, said method comprising:
An inflammatory response wherein the anti-inflammatory agent is induced by the internalizing peptide, comprising administering an anti-inflammatory agent to a subject who has been or will be administered a pharmacological agent linked to the internalizing peptide To inhibit the method.   A method of delivering a pharmacological agent to a subject, the method comprising administering to the subject the pharmacological agent linked to an internalizing peptide, the subject administering an anti-inflammatory agent. A method wherein the anti-inflammatory agent has been or has been administered so that the anti-inflammatory agent inhibits an inflammatory response induced by the internalizing peptide.